Hematopoietic stem cells are rare primitive blood cell progenitors that have the capacity both to self-replicate, so as to maintain a continuous source of regenerative cells, and to differentiate, so as to give rise to various morphologically recognizable precursors of blood cell lineages. These precursors are immature blood cells that cannot self-replicate and must differentiate into mature blood cells including the erythroid, lymphoid and myeloid cells. Within the bone marrow microenvironment, the stem cells self-proliferate and actively maintain continuous production of all mature blood cell lineages throughout life.
Bone marrow (BM) transplantation is being increasingly used in humans as an effective therapy for an increasing number of diseases, including malignancies such as leukemias, lymphoma, myeloma and selected solid tumors as well as nonmalignant conditions such as severe aplastic anemias, immunologic deficiencies and inborn errors of metabolism. The objective of BM transplantation is to provide the host with a healthy stem cell population that will differentiate into mature blood cells that replace deficient or pathologic cell lineages.
The source of the BM for transplantation may be autologous, syngeneic or allogeneic. Preferred are autologous BM or BM from HLA-matched siblings, but also BM from HLA-nonmatched donors is being used for transplantation.
Complicating factors in BM transplantation include graft rejection and graft-vs-host disease (GVHD). Since donor T lymphocytes were found to cause GVHD in animals, one of the procedures to prevent or alleviate GVHD consists in removing T cells from the donor BM before transplantation. This can be done by different techniques, e.g. by soybean agglutination and E-rosetting with sheep red blood cells, or by treatment with anti-T lymphocyte monoclonal antibodies. Extensive use of T-cell depleted BM effectively prevented GVHD but, unfortunately, resulted in a high rate of graft rejection (10–15% in HLA-matched recipients and 50% in HLA-nonmatched recipients) or graft failure (as high as 50%).
Another problem in BM transplantation is the difficulty of achieving long-term successful engraftment also when no graft rejection or GVHD occurs. Nowadays, patients which were successfully transplanted have very low levels of stem cells and immature progenitors which generate mature blood cells, compared with healthy individuals.
Stem cells are functionally defined by their ability to home to the bone marrow and to durably repopulate transplanted recipients with both myeloid and lymphoid cells. The processes that mediate homing and engraftment of human stem cells to the bone marrow involve a complex interplay between cytokines, chemokines and adhesion molecules.
Much of our knowledge of the regulation and the hierarchical organization of the hematopoietic system derives from studies in the mouse wherein stem cells are identified and quantified in long-term reconstitution assays. In contrast, our knowledge of the biology of human hematopoiesis is limited, since it is mostly based on in vitro assays or clinical bone marrow transplantation protocols, both lacking the option to characterize and quantify repopulating stem cells.
Intensive research is being carried out in order to understand the processes that mediate homing and engraftment of human stem cells to the bone marrow. Recently, several groups have established in vivo models for engrafting human stem cells, e.g. into immune deficient mice such as irradiated beige, nude, Xid (X-linked immune deficiency), SCID and non-obese diabetic SCID (NOD/SCID) mice, and in utero transplantation into sheep fetuses, which resulted in successful multilineage engraftment of both myeloid and lymphoid cells (McCune et al., 1988; Nolta et al., 1994; Lapidot et al., 1992; Larochelle et al., 1996; Civin et al., 1996).
Previously, the present inventors have developed a functional in vivo assay for primitive human SCID repopulating cell (SRCs) based on their ability to durably repopulate the bone marrow of intravenously transplanted SCID or NOD/SCID mice with high levels of both myeloid and lymphoid cells (Lapidot et al., 1992; Larochelle et al., 1996). Kinetic experiments demonstrated that only a small fraction of the transplanted cells engrafted and that these cells repopulated the murine bone marrow by extensive proliferation and differentiation. Furthermore, the primitive human cells also retained the capacity to engraft secondary murine recipients (Cashman et al., 1997). Transplantation of populations enriched for CD34 and CD38 cell surface antigen expression, revealed that the phenotype of SRC is CD34+CD38− (Larochelle et al., 1996). Other repopulating cells may exist since recent studies suggest that immature human CD34− cells and more differentiated CD34+CD38+ cells have some limited engraftment potential (Zanjani et al., 1998; Conneally et al., 1997).
Accumulating evidence indicates that stem cell homing to the bone marrow is a multistep process. The mechanisms and specific adhesion molecules involved in this process are not fully understood. The β1 integrins very late antigen 4 (VLA-4) and VLA-5 and the β2 integrin lymphocyte function-associated 1 (LFA-1) have been shown to be implicated in the adhesive interactions of both mouse and human progenitor cells with the bone marrow extracellular matrix (ECM), as well as with bone marrow stromal cells (Levesque et al., 1995). VLA-4 plays an especially important role in murine stem cell migration and hematopoiesis in vivo. Murine stem cells lacking β1 integrins fail to colonize the fetal liver (Hirsh et al., 1996).
Similarly, homing of lymphocytes into lymphoid tissue and migration of leukocytes to inflammation sites are also mediated by adhesion molecules and by an entire family of chemoattractant cytokines (chemokines) and their cell surface receptors. Chemokines are cytokines that are best known for their ability to selectively attract subsets of leukocytes to sites of inflammation. However, chemokines are also important regulators of human development, hematopoiesis and angiogenesis. Activation of chemokine receptors in leukocytes results in a multistep process that includes activation of cell surface adhesion molecules followed by firm adhesion to the vessel wall and eventually migration into the extravascular compartment (Premack and Schall, 1996).
Stromal cell-derived factor 1 (SDF-1) is a noted chemokine also known as pre-B cell growth stimulating factor (Nagasawa et al., 1996). Human and murine SDF-1 differ in one amino acid and are crossreactive. SDF-1 is the ligand for the CXCR4 receptor (previously identified as the orphan chemokine receptor fusin/LESTR), which is expressed on many cell types, including some CD34+CD38− cells (Bleul et al., 1996). In vitro SDF-1 attracts CD34+CXCR4+ cells, and was also shown to induce rapid activation of LFA-1 and VLA-4 on human CD4+ T cells (Aiuti et al., 1997; Campbell et al., 1998). In vivo SDF-1 is produced by bone marrow stromal cells as well as by epithelial cells in many organ (Bleul et al., 1996; Aiuti et al., 1997). Mice that lack SDF-1 or do not express CXCR4 exhibit many defects, including the absence of both lymphoid and myeloid hematopoiesis in the fetal bone marrow (Nagasawa et al., 1996). A defect in stem cell homing to the bone marrow may be one explanation for such a phenotype. Overexpression of human CD4 and CXCR4 receptors on murine CD4+ T cells led to enhanced homing of these cells to the murine bone marrow (Sawada et al., 1998).
In view of the expanded approach to treatment of many severe diseases by hematopoietic stem cell treatment, it is highly desirable to understand better the mechanism behind stem cell horning to the bone marrow and repopulation of transplanted hosts in order to obtain stem cells with higher rates of successful and long-tem engraftment.